Bioaffinity and ion exchange separations with liquid exchange supports

ABSTRACT

Bioaffinity and ion-exchange separation methods are provided along with liquid supports utilized in these methods. The support is based on an inert carrier with ligands or binders attached to its surface through a highly fluorinated isocyanate anchor group. Methods for preparing such supports and their use in capturing neutral and charged target molecules from samples and in analytical applications are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a division of application Ser. No. 07/134,026, filed Dec. 17,1987, now abandoned which is a continuation-in-part of an applicationentitled Bioaffinity Separations with Liquid Affinity Supports, Ser. No.032,642, filed Mar. 31, 1987 which is a continuation-in-part of Ser. No.863,607, filed May 15, 1986 both by Julian P. Breillatt, Jr. et al.

TECHNICAL FIELD

This invention is related to the performance of affinity and ionexchange separations and more specifically to the performance ofbioaffinity and ion exchange separations utilizing perfluorocarbonfluid-based liquid supports and their use in capturing molecules throughspecific binding and ionic reactions.

BACKGROUND ART

An affinity separation can be defined as any separation achieved byemploying the specific binding of one molecule by another. Bioaffinityseparation is defined as an affinity separation in which one of thecomponents involved in the affinity reaction is biologically active oris of biological interest. Bioaffinity separations generally involve atleast one biomacromolecule, such as a protein or nucleic acid, as one ofthe components of the binding pair. Examples of such bioaffinity bindingpairs include: antigen-antibody, substrate-enzyme, effector-enzyme,inhibitor-enzyme, complementary nucleic acid strands, bindingprotein-vitamin, binding protein-nucleic acid; reactive dye-protein,reactive dye-nucleic acid; and others; the terms ligand and binder willbe used to represent the two components in specific bioaffinity bindingpairs. This type of specific binding is distinct from the partitioningof a solute between two solvents. Such partitioning is based onhydrophilicity or hydrophobicity considerations and is relied on, forexample, in solvent extraction, high performance liquid chromatographyand gas liquid chromatography.

Affinity separations are generally considered to require the use ofsolid supports derivatized with a ligand or binder. These separationscan be configured as batch processes or chromatographic processes withthe latter generally being preferred. Affinity chromatography is wellknown and has been reviewed, for example, in C. R. Lowe, "AnIntroduction to Affinity Chromatography". North Holland PublishingCompany, Amsterdam, N.Y. 1978. Lowe describes the characteristicsdesirable in a solid support to be used in an affinity separation.According to Lowe, the solid support should form a loose, porous networkto allow uniform and unimpaired entry and exit of large molecules and toprovide a large surface area for immobilization of the ligand; it shouldbe chemically inert and physically and chemically stable; and thesupport must be capable of functionalization to allow subsequent stablecoupling of the ligand. Additionally, the particles should be uniform,spherical and rigid to ensure good fluid flow characteristics.

The list of support materials suitable for affinity chromatography isextensive and will not be reviewed here (see Lowe, 1978. for a partiallisting). It is not generally possible for a given support to achieveall of the above objectives. One compromise is evident in the use ofhigh surface area porous supports, such as porous glass or porouspolyacrylamide beads. These type supports have the disadvantage thatwashing away contaminating substances is difficult due to thetortuous-paths and dead end channels in these supports (Eveleigh,Journal of Chromatography, Volume 159, 129-145 (1978)]. Anotherdisadvantage of porous supports is that not all of the surface area isusable with large macromolecules. That is, the pore size may be toosmall to allow the macromolecule to enter thereby limiting the effectivesurface area and capacity [Zaborsky, Biomedical Applications ofImmobilized Enzymes and Proteins, Volume 1. p. 41. Ed. Chang, PlenumPress (1977)].

A further practical disadvantage of standard solid support affinitychromatography is the decreasing efficiency of the column as bindingsites at the incoming end of the column become filled with the targetbinder molecules. The binder then must flow further down the column tofind a free ligand and, therefore, the probability of the elution of thetarget prior to binding increases. Countercurrent chromatography wherethe sample is constantly exposed to fresh support overcomes thisdisadvantage. However, affinity supports are not generally amenable tosuch processes because the support is not conveniently pumped as aslurry. Solid supports are ideally suited for use in packed beds (e.g.,columns): they can be packed well, are easily retained on porous fritsand, being rigid, are self supporting. Problems arise, however, when onetries to transport them. They tend to sediment irreversibly in low flowareas, pack together as a mass if obstructed, and the particles abradeby contact with each other and the containing walls. Slurries ofparticles could be transported at low concentrations. That, however, isimpractical requiring transportation of the carrier fluids also. A majorproblem in designing a practical continuous system is how to transport apacked slurry without carrying over fluids from one stage to the next.Seals or gates introduce abrasion and invariably leak or fail by virtueof compaction of sediment within them and fragmentation of the supportrapidly becomes apparent.

Continuous chromatographic separation, using solid supports, has beenmade possible using a rotating annular bed in which sample is applied ata fixed point in a descending curtain of elution fluid, separatedcomponents being collected around the lower periphery. Such devices arecumbersome to construct and operate and suffer from the majordisadvantage that the bulk of the bed (support) is not being utilizedfor the separation. Furthermore, problems associated with an evendistribution of eluant flow, sealing, and optimization of the separationhave inhibited general exceptance of the approach.

A still further disadvantage of solid supports is their propensity tobecome plugged with debris from the sample. This may be cellular debrisfrom a biological sample or physical debris from other sources, butsamples frequently require filtration prior to processing in order topreserve the good flow characteristics of the column.

Affinity separations often form a component part of other processes. Oneexample is their use in heterogeneous immunoassays. Here the affinityseparation is used to capture an analyte from a complex mixture such asserum or plasma. After capturing the analyte, the contaminants arewashed away and the analyte is detected using any number of well knownassay protocols.

Some common solid supports in this area are plastic spheres (beads),interiors of plastic test tubes, interiors of microtitre plate wells,magnetic particles, and porous glass particles. The largest disadvantageof these systems is the generally limited surface area which limitscapacity and capture efficiency. This, in turn, leads to a limitation insensitivity (change in response/change in concentration) and detectionlimit (minimum detectable concentration).

Certain separation problems have been traditionally dealt with byliquid-liquid extractions. For example, in nucleic acid hybridizationassays, requiring purified nucleic acid. a nucleic acid from the sample,such as DNA or RNA, needs to be bound to a solid support. To obtain thenucleic acid to be probed it must first be released from a cell (ifwithin a cell), by lysis, then extracted from the lysate. The mostcommon extraction technique uses an aqueous phenol/chloroform mixture(Maniatis et al., Molecular Cloning: A Laboratory Manual, pp. 458-9,Cold Spring Harbor Laboratory, 1982). Proteins, which are the majorcomponent of the lysate, tend to interfere with the extraction.Following extraction of the nucleic acid, excess phenol must beextracted with ether and then the ether evaporated. The nucleic acidcontaining solution is then concentrated prior to deposition on a solidsupport; see, for example, Church et al. Proc. Nat. Acad. Sci. USA,Volume 81, 1991 (1984). This is a tedious and hazardous process withmany opportunities for material losses along the way.

Some liquid phase affinity partitioning separations have been achieved.P-A Albertsson, "Partition of Cell Particles and Macromolecules",Almquist and Wiksell, Ed.; Wiley, New York, 1971, reported thedevelopment of a partitioning system based on the immiscibility ofaqueous solutions of dextran and polyethylene glycol. S. D. Flanagan etal., Croatian Chem. Acta, Volume 47, 449 (1975), adapted that system toallow affinity mediated separations by attaching ligands to thepolyethylene glycol thus allowing specific binding affinities to drivethe separation. This system has limited utility in that it requires thatthe binder first partition into the phase containing the ligand to somedegree before the specific binding interaction can occur. The system isfurther limited in that it is applicable only to batch processes and notto chromatographic processes.

Perfluorocarbon emulsions have been used to study cell-substrateinteractions as reported by Keese et al. [Proc. Nat. Acad. Sci. USA,Volume 80, 5622-5626 (1983)]. While anchorage-dependent cells aretraditionally grown on solid supports. Keese et al. have shown that theycan be grown at the phase boundary between liquid perfluorocarbons andtissue culture medium. Keese et al. showed that surface active compoundssuch as pentafluorobenzoyl chloride provided an effective surface forgrowing such cells. However, because of evidence indicating no reactionbetween the acid halide and the cells or proteins present in the culturemedium, the authors speculated that the pentafluorobenzoyl chloride washydrolyzed to form pentafluorobenzoic acid on the surface of theperfluorocarbon droplet. The authors further speculated that the acidsurface caused a layer of denatured protein to deposit on the surface ofthe liquid perfluorocarbon providing a suitable surface for attachmentof the cells. As further proof of the lack of acid chloride-amino groupreactions, the authors obtained cell attachment and proliferation bysonicating the emulsion, without perfluorobenzoyl chloride, with wateror ethylene glycol. Keese et al. speculated that unknown surface activecompounds were being formed by this treatment.

Because affinity separation is a powerful technique and becausecurrently available supports suffer from various disadvantages, there isa need for improved supports. These should have the followingproperties: physical and chemical stability; chemical inertness;compatibility with a variety of biological samples; utility in batch andchromatographic applications and in countercurrent type applications;high surface area; ability to allow high flow rates in chromatographicapplications; ability to provide for ready and stable attachment ofligands or binders to the surface; allow simple concentration of thecaptured product; allow easy automation of any separation process; andallow simple efficient regeneration of the support.

DISCLOSURE OF THE INVENTION

The liquid supports of this invention are based on liquidperfluorocarbon carriers to which ligands or binders are securelyattached through a highly fluorinated isocyanate anchor group. Theliquid supports are chemically inert, immiscible with water, havespecific gravity significantly different from water, and have lownonspecific binding to the ligands and binders.

The method of conducting bioaffinity separations comprising the stepsof:

1) forming a liquid support by attaching a ligand or binder to thesurface of the droplets of an emulsion of a liquid perfluorocarboncarrier through a highly fluorinated isocyanate anchor group; and

2) capturing a target binder or ligand, complementary to the ligand orbinder attached to the carrier from a mixture using said liquid support.

The ligand or binder is attached to the surface through modification ofthe surface or of the ligand or binder by the direct or the partitionmethod utilizing a highly fluorinated isocyanate anchor group based on acompound having the formula:

    R.sub.F --CH.sub.2 CH.sub.2 CH.sub.2 --NCO

wherein R_(F) is linear, branched, or cyclic perfluorinated radical from1 to 20 carbon atoms.

DISCUSSION OF THE INVENTION

The liquid supports of this invention offer unprecedented advantages incarrying out bioaffinity separations. Since the liquid supports of thisinvention, in addition to being liquid affinity supports also includesuch separations which rely on charged interactions, e.g. when theligand attached to ths carrier is a charged organic moiety and theseparation of the desired product from its environment is by the processcommonly referred to as ion exchange separation (chromatography), thebioaffinity separations of this invention are intended to include suchion exchange separations and the liquid affinity supports are intendedto include liquid ion-exchange supports. Two of the greatest advantagesof using a liquid affinity support are: allowing development ofcountercurrent affinity separations and allowing simple concentration ofthe captured product. Other advantages are allowing easy automation ofthe separation process and allowing high flow rates in chromatographicapplications. The liquid supports of this invention offer additionalproperties such as being stable in an aqueous environment; beingimmiscible with water; having a specific gravity significantly differentfrom water, preferably heavier, allowing easy separation from theaqueous environment; having low nonspecific binding to native proteins,nucleic acids or other components of biological samples; and beingincapable of dissolving any extraneous solutes present in a biologicalsample.

The liquid affinity supports of this invention comprise a carrier andligands or binders which are securely attached through a highlyfluorinated isocyanate anchor. Carriers useful in carrying outbioaffinity separations have the properties described above and includeliquid perfluorocarbons (LPF), hydrocarbons, and silicones. Byperfluorocarbon is meant a molecule which contains the largest possibleor a relatively large proportion of fluorine atoms in its structure.

LPF's are known to be inert and biocompatible. Liquid perfluorocarbonshave been used as blood substitutes in the form of emulsions ofperfluorocarbons in isotonic buffers containing antibiotics [Agarwal,Defense Science Journal, Volume 30, 51-54 (1980)). This use depends onthe high solubility of oxygen in these emulsions and does not requireany surface modification of the perfluorocarbon droplets. It does,however, require the presence of surfactants such as phospholipids tostabilize the emulsion. LPF's are immiscible with water and are verypoor solvents for both hydrophilic and hydrophobic solutes. As a class,LPF's are dense liquids with specific gravities (approximately 2)allowing simple gravity separation of the support from the samplematrix.

Hydrocarbons and silicones are other classes of materials which meet therequirements to function as carriers in liquid affinity supports.

By ligand is meant an antigen, hapten, nucleic acid, enzyme substrate,vitamin, dye, charged organic molecules or other small organic moleculeincluding enzyme effectors and inhibitors and by binder is meant anantibody, enzyme, nucleic acid, binding protein, synthetic mimics ofbinding proteins such as polylysine and polyethyleneimines or otherbiomacromolecule capable of specific binding or ionic (charged)interactions.

The highly fluorinated isocyanate anchor group is based on a compoundhaving the formula R_(F) CH₂ CH₂ CH₂ NCO, wherein R_(F) is linear,branched, or cyclic perfluorinated radical from 1 to 20 carbon atoms. Ina preferred class of the above compounds R_(F) is a linear F(CF₂)_(n)radical. A more specifically preferred compound is F(CF₂)₈ CH₂ CH₂ CH₂NCO. These compounds can be made in the following manner. A startingfluorinated olefin, R_(F) --CH═CH₂, is contacted with HCN in thepresence of a nickel catalyst and a Lewis acid promoter such as zincchloride by a process similar to that disclosed in Drinkard et al., U.S.Pat. No. 3,496,217. By using conventional chemistry, the resultingnitrite, R_(F) --CH₂ CH₂ --CN, is converted to the amine, R_(F) --CH₂CH₂ CH₂ --NH₂, by a reaction with hydrogen in the presence of ammoniaand Raney cobalt. The isocyanate, R_(F) --CH₂ CH₂ CH₂ --N═C═ O, then canbe prepared by the reaction of the amine with phosgene.

The affinity support must have the ligand or binder securely attached tothe carrier. By secure attachment is meant an attachment capable ofsurviving the steps involved in the bioaffinity and ion exchangeseparations while permitting two-dimensional mobility of the ligand onthe carrier surface. However, it is expected that this attachment needsto be reversible when desired, for example, during the concentrationstep leading to a ligand-binder conjugate, such as by evaporation, andwhen desiring to regenerate the carrier, such as by competitivedisplacement of ligand or binder by perfluoro-surfactants. This isnecessary so that ligand or binder does not contaminate the purifiedproduct and also to prevent loss of capacity of the support. With solidsupports this is usually accomplished by covalently attaching the ligandor binder to the support. In addition to attaching securely ligand orbinder, it is desirable not to alter the general inertness of thecarrier nor introduce functional groups which might increase nonspecificbinding. Further, it is desirable to develop general methods which willbe applicable to a variety of ligands or binders.

One method for attaching ligands or binders to the surface of thecarrier droplets to prepare a liquid affinity support is referred to asthe direct method. In this method, an LPF containing a soluble reactiveperfluorinated compound is emulsified with an aqueous solution of theligand or binder. Compounds which are soluble in the LPF are generallythose containing a high proportion of fluorine atoms. Commerciallyavailable perfluoroalkyl and perfluoroaryl compounds can be used.Suitable reactive groups are well known to those skilled in affinitychromatography; those reactive with amino groups being preferred.Examples of appropriate anchor compounds include pentafluorobenzoylchloride, perfluorooctanoyl chloride, and perfluorooctanoyl anhydride,and perfluorooctylpropyl isocyanate. This isocyanate is a member of aclass of compounds represented by the formula R_(F) (CH₂)₃ NCO, whereinR_(F) is as defined above. These compounds are described in applicants,assignee's copending application, filed on even date herewith,incorporated hereby by reference. The desired ligand or binder inaqueous solution is then emulsified with the carrier containing thereactive compound to permit the reaction of the reactive group withamino groups (or other suitable functional groups) on the ligand orbinder.

It has been found that the amount of perfluorinated reagent on thesurface of the droplet is important in obtaining an optimal layer ofligand or binder, for example, a protein. Too much reagent causes thedeposition of very thick protein layers resulting in distorted and shearsensitive droplets. Further, thick coatings result in inefficient usageof the ligand or binder. Too little reagent results in low capacitysupports which are also not desirable. The optimization of the amount ofperfluorinated reactive reagent in this application is analogous tooptimization of coupling reagents commonly used with solid supports andis well known to those skilled in preparing solid affinity supports.Among factors which are important in this process are the susceptibilityof the reactive reagent to hydrolysis, the pH of the reaction mixtureand the time of exposure.

The second and preferred method for preparing liquid affinity supportsis referred to as the partition method. The basic difference betweenthis and the direct method is that in the partition method the ligand orbinder is modified to permit its selective high affinity (secure)binding to the surface of the carrier droplets. One means to accomplishthis is to prepare and purify reagents, which contain a highly orperfluoro-substituted ligand or binder, prior to attachment to thesurface. Several well known chemical strategies can be used to attachcovalently highly fluorinated groups to ligands or binders. Factorswhich should be considered are reactivity of the fluorinated compoundused, the pH of the reaction medium, the time and temperature of thereaction.

Compounds such as isocyanates, acid chlorides, anhydrides andimidazolides of various perfluorocarbon acids, for example,perfluorooctylpropyl isocyanate, perfluorooctanoyl chloride,perfluorooctyl acetyl and propanoyl chlorides and perfluorooctanoyl andperfluorooctyl propanoyl imidazolides have been used successfully duringthe preparation of liquid affinity supports of this invention. Theimidazolide derivative is preferred due to its lower reactivity allowingmore controllable reactions. The most preferred class of compounds isthe highly fluorinated isocyanates, as described above, an example beingperfluorooctylpropyl isocyanate. The isocyanates are most preferredbecause of their increased stability to hydrolysis at the slightlyalkaline reaction conditions generally used during the preparation ofthe liquid supports of this invention. In addition, the small amount ofhydrolysis products formed (amines and ureas) do not interfere with theadsorption of the perfluoro-modified ligand or binder to the surface ofLPF droplets when the partition method is used to form the liquidsupports of this invention. This eliminates the need to purify theperfluoro-modified ligand or binder from the perfluoro-modificationreaction milieu prior to forming the liquid support.

In general, the reactions are carried out by mixing an aqueous solutionof the ligand or binder with the fluorinated reagent dissolved in awater miscible organic solvent such as tetrahydrofuran under controlledtime, temperature and pH conditions. The derivatized ligand or bindercan be separated from the by-products of the reaction and the organicsolvent by gel filtration or dialysis. The degree of derivatization canbe determined by any of the known techniques such as trinitrobenzenesulfonate labeling. The substituted ligand or binder is now ready to beused to form the liquid affinity support with an appropriate carriersuch as a specific LPF. The liquid affinity support is formed byemulsifying the LPF such as perfluorodecalin with a buffered solution ofthe derivatized ligand or binder which partitions onto the surface ofthe forming droplets. The ligand or binder also acts as the emulsifyingagent (surfactant) and the adsorbed layer prevents re-coalescence.

The degree of derivatization (substitution) required to provide secureattachment to the surface of the droplets is expected to varysignificantly depending upon the nature of the perfluoro anchor group,the spatial arrangement of the anchor groups on the ligand, the size andnature of the ligand, and the eventual use of the liquid support. Ingeneral, the higher the degree of substitution the stronger theattachment. This, however, can be limited by steric considerations aswell as the need to retain the biological activity of the ligand orbinder. It has been found that placing anchor groups on approximately20% of the available amino groups on a typical protein is preferred.When 20% of the amino groups of glucose oxidase and urease were labeledwith perfluorooctyl imidazolide, these enzymes were found to retainsubstantially all of their native activity. Also, when washed withbuffers, the enzymes resisted being washed off the surface ofperfluorodecalin droplets.

A third method for preparing liquid supports is a variation of thepartition method. In this approach, a reagent comprised of a highly orperfluoro-substituted moiety and a charged organic moiety is utilized.The highly or perfluoro-substituted portion acts as a hydrophobic moietywhile the charged organic portion acts as a hydrophile, constituting acharged (ionic) surfactant. This water soluble or water misciblesurfactant can be mixed with, for example, an LPF. The result is thesecure attachment of the charged organic molecule (moiety) to thesurface of the carrier droplets trhough the fluoro-substituted portion.In the laboratory, mixing can be carried out in a separatory funnel orflask; small quantities of liquid supports can be prepared by agitationwith a vortex mixer, in general, after the formation of the liquidion-exchange support, the reaction mixture can be washed with water oran aqueous buffer solution to remove excess fluoro-surfactant. Theincorporated surfactant imparts stability to the emulsion while thecharged portion, the ligand, imparts selectivity when the supports areused in separation and extraction procedures.

Charged organic molecules having fluoro-substituted portions include aclass of surfactants available under the name of Zonyl®fluorosurfactants (a registered trademark of E. I. du Pont de Nemoursand Company). These include Zonyl® FSP, FSE phosphate salts, [F(CF₂CF₂)₃₋₈ CH₂ CH₂ O]₁.2 P(O) (O⁻ NH₄ ⁺ 2.1; Zonyl® FSC sulfate salts,F(CF₂ CF₂)₃₋₈ CH₂ CH₂ SCH₂ CH₂ N⁺ (CH₃)₃ ⁻ OSO₂ OCH₃ ; etc.

While the approaches described here provide good general procedures forattaching ligands or binders to the surface of the carriers,specific-procedures for specific ligands or binders may need to beutilized. One such procedure would involve specific substitution of theFc portion of an IgG class antibody with a highly fluorinated reagentallowing the attachment of the antibody to the surface of the LPF in aspecific orientation. This would allow attachment of the antibody withits specific binding portions, the F(ab) binding sites, oriented intothe aqueous portion of the emulsion. Such orientation is expected toprovide more efficient use of the antibody and greater captureefficiency. It might also minimize nonspecific binding interferences byrheumatoid factors which might be present in the mixture by making theFc portion of the antibody inaccessible to the aqueous phase.

While both of the above-described methods can be utilized in the instantinvention, the partition method has some advantages over the directmethod. These advantages include providing for preparation of individualcomponents of the liquid affinity support permitting more rigorousquality control; promoting optimal use of expensive or scarce ligands orbinders; creating a single ligand layer minimizing steric blockage ofbinding sites on the support; and providing multiple attachment sites oneach ligand or binder promoting stronger attachment to the surface ofthe droplet.

The two-dimensional mobility of the ligand or binder inherent in theliquid affinity support may offer a further advantage, particularly withlarge macromolecules such as DNA, by allowing a zippering effect tooccur. By zippering effect is meant the successive binding of variousbinding sites of the target molecule to mobile binding sites on thedroplet surface. Such binding to each successive site of the carrierpromotes alignment of the DNA with and attachment to the next bindingsite of the carrier. This is expected to provide secure, shear resistantcapture of large and potentially fragile macromolecules. This zipperingeffect would not be possible with solid affinity supports.

Another advantage of liquid supports over conventional solid supports isthe ability to sterilize the reagents used to form the support as wellas to re-sterilize contaminated supports. The latter is not possiblewith solid supports. The partition method of attaching ligands orbinders to the carrier is particularly amenable to re-sterilization. Theperfluoro-derivatized ligand or binder can be recovered from the supportusing perfluorosurfactants and sterilized by ultrafiltration prior toreattachment.

The LPF's can also be sterilized by ultrafiltration or autoclaving. Thesupport can then be reformed using the same components or fresh LPFcould be substituted. This allows recovery and reuse of valuable ligandor binder. Certain supports can also be sterilized without separation ofthe components if the ligand or binder can retain biological activityunder appropriate sterilization conditions. These considerations areparticularly important to applications such as extra-corporeal bloodprocessing or preparation of therapeutics for use in humans.

The use of the liquid affinity supports described above can be quitedifferent from the use of conventional solid affinity supports. Liquidaffinity supports offer a choice of separation modes. While solidsupports are generally limited to concurrent operation, liquid supportscan operate in the countercurrent or in interconnected multiple batchmodes. This flexibility allows the development of separation processesthat are governed by the needs and contraints of the overall systemrather than the need to use a particular separation method. Theseparated material can be recovered from the liquid affinity supportthrough the use of dissociation agents.

In chromatographic applications, solid affinity supports usually requirethe use of filtered samples to prevent debris from occluding the columncausing high back pressures, decreased fluid flow and generally lessefficient operation. Liquid affinity supports, by their inherentdeformability, resist occlusion. The inherent properties ofdeformability, flexibility and instant shape recovery further allowthese supports to be readily transported through tubes without concernfor blockage or physical attrition of the support which occur with solidsupports. Even emulsions containing high concentrations of a liquidaffinity support can be transported with ease.

The ability to be transported conveniently through tubes provides forready automation of the separation process, only standard pumps andvalves are required and, in many circumstances, even solenoid valves canbe avoided as a high concentration emulsion can effectively act as avalve. At a restricted orifice, the dense LPF-based slurry can resistpassage through the orifice and prevent passage of the aqueous streamwhile allowing controlled passage of the emulsion. The flow of emulsioncan be readily controlled by the dimension of the orifice and therelative fluidic pressures across this constriction.

Use of liquid affinity supports in the batch mode can also offeradvantages over solid affinity supports; the separation from the aqueousphase is rapid and simple because of the high density of the support;washing of the support is simple and efficient because the support isnon-porous, and the difficulties of porous supports or the fragility ofextremely small particles are avoided while taking advantage of the highsurface area of the support.

The bioaffinity separation method of this invention permits theutilization of countercurrent affinity separation not practicable withstandard solid phase affinity supports because the latter cannot betransported reproducibly and consistently. Such separations offer themaximum capture efficiency because the leading front of the samplestream which is most depleted in the target ligand or binder isconstantly exposed to fresh affinity support which has the maximalability to capture the remaining target molecules. This can be used togreat advantage to collect a dilute product from a process stream,particularly when used in a continuous process. Another advantageous useof these separations is in depleting a sample stream of deleterioussubstances such as waste products as has to be done in extra-corporealplasma depletion.

The method of this invention can also be utilized in the so-called mixand sediment process. Here, the sample stream and liquid support arebrought together in a mixing area which allows the target substance tobe captured by the support. The resulting emulsion is then fed into asedimentation tank where the dense affinity support settles and isdrained for eventual recovery of the target substance and regenerationof the support. This mix and sediment process offers a simple approachto continuous affinity separation.

One particular analytical application of the liquid affinity supports ofthis invention is their use to capture DNA from solution. The partitionmethod was used to form a liquid affinity support with histone proteinsattached to the surface of the droplets. Histones are highly positivelycharged proteins which interact with DNA in the cell to package the DNAinto a compact form. Surprisingly, it has been found that a liquidaffinity support with modified calf thymus histones on the surface ofperfluoro-2-butyltetrahydrofuran or other carriers can capture DNA fromaqueous solution. The captured DNA-support complex can be concentratedby evaporation to afford a histone-DNA complex which is permitted toattach to a suitable membrane. Again, surprisingly, this form of the DNAwas found suitable for hybridization with an appropriate probe. Thisapproach is superior to the current liquid-liquid extraction or columnelution methods both of which require concentration of the DNA afterisolation from solution before it can be applied to the hybridizationmembrane. While histones were used in the described process otherligands, such as polylysine, anti-DNA antibodies or specificoligonucleotide sequences which would capture only complementary basesequences, can also be utilized.

Another analytical application which illustrates the advantages of theuse of liquid affinity supports, is the immunoassay. One such assay is aqualitative enzyme linked immunosorbent assay (ELISA) in which color canbe visually detected on the surface of filter paper, porous membrane,plastic paddle or other solid surfaces. This assay can be readilyadapted to quantitative assays and to the use of other detectablesignals besides color. In the traditional format of ELISA, the use ofporous supports permits the colored product of the immunoassay to betrapped inside the pores diminishing its contribution to the detectablesignal. Also, the colored product often diffuses into solution furtherdiminishing the detectable signal limiting the apparent sensitivity ofthe assay. The apparent sensitivity of these assays can be improved byusing liquid affinity supports of this invention and evaporating thesupport at appropriate stages of the assay. This could be after theanalyte has been captured, analogous to the system described for the DNAassay above, or just before addition of the enzyme substrate to beconverted into colored product.

An alternative to evaporating the liquid support is the release of thebound target substance from the ligand or binder. In the case ofcapturing DNA using modified calf histones, such release can beaccomplished by adjusting the salt concentration of the buffers used towash the support. In the case of an antigen capture on an antibodycoated support, this might be done utilizing a high pH buffer, mildchaotropic agent or fluorosurfactants.

The liquid ion-exchange supports of this invention have utility similarto that described above for the liquid affinity supports but operate onthe principles of ion-exchange.

The following examples further illustrate the invention.

EXAMPLE 1 PREPARATION OF A LIQUID AFFINITY SUPPORT-DIRECT METHOD

Fluorescently labeled bovine serum albumin (FITC-BSA) was prepared byadding 25 mg of fluorescein isothiocyanate (FITC) to 500 mg of bovineserum albumin (BSA) dissolved in 25 mL of 0.2M carbonatebicarbonatebuffer, pH 9.25. The reaction mixture was stirred for 30 minutes at roomtemperature and applied to a 25×2.2 cm column of Bio-Gel® P-6 (Bio-RadLaboratories, Richmond, Calif.) equilibrated with 0.9% sodium chloride(normal saline). The FITC-BSA was eluted in the exclusion volume of thecolumn, free of unreacted FITC. Spectroscopic analysis of the FITC-BSAshowed that the degree of conjugation was 8.65 moles of FITC per mole ofBSA.

Solutions of (perfluorooctyl) acetyl chloride, 1H,1H-perfluorodecanoylchloride (Riedel-De-Haenag, Seelze-Hannover, West Germany) inperfluorodecalin (Flutec PP5. Medical Grade, ISC Chemicals Ltd.,Avonmouth, UK) were prepared as follows. A concentrated stock solutionwas prepared by adding 2 μL of the acid chloride to 1 mL ofperfluorodecalin. Working solutions of 0, 1, 5, 10, 20 and 40 Kg of theacid chloride/ml of perfluorodecalin were prepared by adding 0, 0.25,1.25, 2.5, 5 and 10 μL of the stock solution to 1 mL ofperfluorodecalin. A density of 2 g/mL was assumed for the acid chloridewhen calculating the volumes required.

Emulsions of the liquid affinity support were prepared by dropwiseaddition of 0.1 mL of each of the working solutions prepared above into1.0 mL of a vortexing solution of 1 mg/ml FITC-BSA in normal saline.After vortexing for 10-15 seconds, the emulsions were gently mixed forabout 30 minutes on an oscillating mixer and washed by sedimentationwith 1 mL of normal saline solution to remove unbound FITC-BSA. Thewashing was repeated twice with 1 mL each of normal saline solution.

The emulsions were examined under a fluorescence microscope for thepresence of fluorescence associated with the surface of theperfluorodecalin droplets. The amount of fluorescence was greater withthe higher concentrations of acidchloride used. At the 20 and 40 μg/mLconcentrations of the acid chloride, some distortion of the droplets wasevident. When the control working solution, that is, perfluorodecalinwithout acid chloride, was used, only faint fluorescence was observed.This demonstrated that there was only minimal nonspecific adsorption tothe surface of the droplets and, most importantly, that no liquidaffinity support was obtained when the methods of this invention forpreparing these supports were not employed.

EXAMPLE 2 PREPARATION OF LIQUID AFFINITY SUPPORTS-DIRECT METHOD

FITC-BSA was prepared as described in Example 1. In a series ofexperiments, substituting different liquid perfluorocarbons forperfluorodecalin and different reactive compounds for (perfluorooctyl)acetyl chloride in the procedure of Example 1, nine additional supportswere prepared. The liquid perfluorocarbons were perfluorotributylamine,perfluoro(tetradecahydrophanthralene) andperfluoro-2-butyltetrahydrofuran (all from SCM Chemicals, Gainesville,Fla.). The reactive compounds were perfluorooctanoyl chloride,perfluorooctanoic anhydride, and perfluorobenzoyl chloride (SCMChemicals, Gainesville, Fla.).

Emulsions prepared from all combinations of these components yieldedbright fluorescent layers when observed under fluorescence microscope.Control experiments in which the reactive compounds were not included,showed only faint fluorescence. This demonstrated that a variety ofliquid perfluorocarbons and a variety of reactive perfluorinatedcompounds could be used to attach a ligand to a carrier by the directmethod.

When perfluorosuccinyl chloride, a difunctional acid chloride, was usedas the reactive compound, no emulsions could be obtained; rathercrosslinked, gel-like materials resulted.

EXAMPLE 3 ANTIBODY CAPTURE BY LIQUID AFFINITY SUPPORT

A stock solution was prepared by adding 2.0 μL of perfluorooctanoylchloride to 1 mL of perfluorotributylamine. A working solution wasprepared by diluting 10 μL of the stock solution with 1 mL ofperfluorotributylamine. 0.1 mL of this working solution was addeddropwise to a vortexing solution of 1 mg/ml rabbit immunoglobulin G innormal saline. After vortexing for about 15 seconds, the emulsion wasgently mixed for 30 minutes at room temperature. The emulsion was washedthree times with 1 mL each of normal saline solution.

Most of the supernatant liquid was removed and 50 μL of 1.5 mg/mlFITC-labeled affinity purified goat anti-rabbit-IgG antibody was mixedwith the emulsion of the liquid affinity support for 10 minutes at roomtemperature. The emulsion was then washed 3 times with 1 mL each ofnormal saline solution.

In an identical parallel experiment, normal saline wash solutions werereplaced by wash solutions containing 1% BSA. Both resulting emulsionswere examined under fluorescence microscope and were found to havebright, even fluorescent layers coating the droplets. This demonstratedthat when the ligand attached to a carrier was rabbit IgG it was stillimmunochemically recognizable by an appropriate antibody. Since nodifference was noted between the samples washed with or without BSA inthe wash solution, there appeared to have been little or no nonspecificcapture of the FITC-labeled antibody.

EXAMPLE 4 PREPARATION AND USE OF A LIQUID AFFINITY SUPPORT-PARTITIONMETHOD

A. (Perfluorooctyl) propanoyl imidazolide,1H,1H,2H,2H-perfluoroundecanoic imidazolide, was prepared fromperfluorooctyl propanoic acid as follows: 4.9 g of(perfluorooctyl)propanoic acid was dissolved in 15 mL of dry THF andadded to a stirred solution of 1.8 g of carbonyldiimidazole in 35 mL ofdry THF at room temperature. The reaction mixture was stirred for 30minutes, during which time the product began to crystallize. The mixturewas cooled in ice-water and filtered in a glass-fritted filter funnel.The crystals were washed with about 50 mL of ice-cold THF and dried witha stream of dry air. The yield of (perfluorooctyl)propanoyl imidazolidewas 3.8 g. 68% of the theoretical yield. The melting point was 128° C.

6.0 mL of a 1 mg/ml solution of affinity purified goat anti-human IgG(Tago, Burlingame, Calif.) in phosphate buffer, pH 8.0 was cooled in anice bath. 0.6 mL of a 20 mg/ml solution of (perfluorooctyl)propanoylimidazolide, prepared as above, in THF was added dropwise to the cooledsolution with stirring. The reaction was allowed to proceed for 2 hourswith continuous stirring. The reaction mixture was applied to a 3×26 cmcolumn of Bio-Gelf® P-6 (Bio-Rad Laboratories, Richmond, Calif.)equilibrated with phosphate buffer, pH 8.0. The perfluoroalkylatedantibody was eluted in the void volume of the column and collected inabout 21 mL total volume. Its concentration was determinedspectroscopically (no correction for derivatization was used) and wasfound to be about 0.17 mg/ml. The degree of reaction was determined asfollows: The reaction mixture was analyzed for remaining amino groups bystandard procedures using trinitrobenzene sulfonic acid. The amount ofanchor group attached to the IgG was calculated from the difference inthe amount of available amino groups present between the control (noimidazolide) and the preparations containing the imidazolide. Thepercent of the available amines reacted was 25%.

B. 0.25 mL of perfluorodecalin was added to 5.0 mL of a 0.10 mg/mlsolution of (perfluorooctyl)propanoyl substituted goat anti-human IgG,prepared as in Step (A) above, in phosphate buffer, pH 8.0, whilevortexing. The mixture was vortexed for 1 minute and then washed 3 timeseach with 3 mL of 0.1% aqueous BSA.

The immunochemical reactivity and capacity of the thus prepared liquidaffinity support having goat anti-human IgG bound to the surface of theemulsion droplets was measured as follows: 3.2 mL of a 326 μg/mLsolution of FITC-IGG (FITC labeled human IgG was prepared by theprocedure of Example 1 and found to have 2.56 moles of FITC per mole ofIgG) in 0.1% aqueous BSA was dispensed into quartz covets suitable forfluorescence measurements. To the covet was added a small magneticstirring bar and 50 μL of the support. The covet was stirred for 1minute, allowed to sediment for 10 minutes and the fluorescenceintensity of the supernatant measured in situ. This exact procedure wasrepeated 11 times, then repeated with the mixing time extended to 5minutes and finally reapeated with the mixing time extended to 30minutes. Care was taken during this process not to expose excessivelythe reaction mixture to light. The fluorescence intensity of thesupernatant decreased steadily throughout this process indicatingremoval of the FITC-labeled IgG from solution by the support. Thisprocedure was repeated with 3.2 mL each of 163, 82 and 33 μg/mLsolutions of FITC-IGG. The results are summarized in the table below andindicate that the approximate capacity of 50 μL of this support for thisparticular FITC-IGG is 121 μg.

                  TABLE                                                           ______________________________________                                                         FITC-IgG BOUND TO                                            FITC-IgG ADDED (μg)                                                                         SUPPORT (μg)                                              ______________________________________                                        1043             121                                                          522              121                                                          262               84                                                          106               53                                                          ______________________________________                                    

EXAMPLE 5 DNA EXTRACTION

A liquid affinity support based on perfluorodecalin and histone wasprepared using a slight modification of the method described inExample 1. A 100 μg/mL solution of perfluorobenzoyl chloride inperfluorodecalin was prepared and 0.1 mL of this solution was added to 1mL of a vortexing 1 mg/ml solution of calf thymus histones in phosphatebuffered saline to obtain the support.

A digest of the plasmid pBR322 was made according to standard proceduresusing the restriction enzyme Hae II. Acceptable digestion wasdemonstrated by end labelling with radioactive phosphorus labelled ATPusing terminal deoxynucleotidyl transferase followed by electrophoresisand autoradiography to detect the fragments. 13 distinct radioactivefragments were observed indicating acceptable digestion.

20 μL of the pBR322 digest in 10 mM tris buffer, pH 8.0, was mixed with180 μL of 10 mm tris buffer, pH 8.0. containing 1 mm EDTA. A 5-μL samplewas removed to serve as a control. To extract the DNA, 50 μL of thesedimented support was added to the remainder and the mixture rockedgently for 30 minutes. After sedimentation, a 5-μL sample of thesupernatant was removed and the remaining supernatant was transferred toanother tube. The extraction process was repeated four more times withfresh support emulsion.

The supernatant samples were analyzed by electrophoresis on a 6%acrylamide, 7M urea gel using standard procedures. The gel was dried andautoradiographed on Kodak XAR-5 film for about 48 hours and the filmdeveloped in a commercial processor. Examination of the autoradiographshowed that a significant quantity of the labelled nucleic acids hadbeen adsorbed by the histone coated support. The higher molecular weightspecies were adsorbed while the low molecular weight species remained insolution. By comparing the electrophoretic mobility of theoligonucleotides to that of known molecular weight standards, it wasestimated that oligonucleotides containing greater than 21 pairs wereextracted preferentially from the mixture.

EXAMPLE 6 DNA EXTRACTION AND HYBRIDIZATION

A 20 μg/mL solution of digested pBR322 prepared as in Example 5 wasprepared by serial dilution of a 20 mg/ml solution in 10 mm Tris buffer,pH 8.0, containing 1 mm EDTA. 100 μL of this solution was added to 100μL of 25 mM phosphate buffer containing 0.9% sodium chloride, 0.1% BSA,pH 7.8, in a test tube. To this was added 50 μL of a liquid affinitysupport based on perfluorodecalin and histone prepared as in Example 5.The tubes were capped and rocked gently for 60 minutes at roomtemperature. After sedimentation, the supernatant was discarded and theresulting support-DNA complex was washed with 0.5 mL of 25 mM phosphatebuffer containing 0.9% sodium chloride, pH 7.8. The complex wastransferred to a known position on Genescreen+™ hybridization membraneretained in a Hybri-slot apparatus (Bethesda Research Laboratories,Inc., Bethesda, Md.) in several consecutive 10-20 μL portions. Theliquid perfluorocarbon carrier was allowed to evaporate betweenadditions. A 100-μL sample of the original solution of the digest wasdispensed onto other portions of the membrane. The air-dried membranewas further treated to denature, neutralize and prehybridize the nucleicacids bound to the membrane.

The membrane was hybridized with an excess of labelled pBR322 at 65° C.overnight. (Undigested pBR322 was labelled with ³² phosporous using astandard nick translation system.) The membrane was washed, dried, andthe dried membrane was exposed to Kodak XAR-5 film overnight. The filmwas developed in a commercial processor.

This complete procedure was repeated with 2 μg/ml, 200 ng/ml, 20 ng/ml,2 ng/ml and 200 pg/ml, respectively, concentrations of the originalpBR322 digest. The table below summarizes the results of the visualinspection of the autoradiograph of all samples.

                  TABLE                                                           ______________________________________                                        pBR322 DIGEST                                                                              UNPROCESSED  SUPPORT-                                            CONCENTRATION                                                                              DIGEST       DNA COMPLEX                                         ______________________________________                                         20 μg/mL strong       strong                                               2 μg/mL  strong       strong                                              200 ng/mL    strong       strong                                               20 ng/mL    strong       strong                                               2 ng/mL     distinct     strong                                              200 pg/mL    very faint   distinct                                            ______________________________________                                    

These results indicate that DNA captured by binding to a liquid affinitysupport of this invention is still capable of being hybridized and thatthe sample can be concentrated by this procedure. This latter point isdemonstrated especially by the two low concentration samples.

EXAMPLE 7 DNA EXTRACTION AND RECOVERY

A 10 μg/mL solution of Strain B E. coli DNA (Sigma Chemical Co., St.Louis, Mo.) was prepared by dissolving 10 μg in 1 mL of a buffercontaining 20 mM tris, 10 mM sodium chloride, 1 mM EDTA, pH 8.0. in atest tube. To this solution was added 150 μL of a liquid affinitysupport based on histone, prepared as in Example 5. The tube was cappedand rocked gently for 60 minutes at room temperature. Aftersedimentation the supernatant was removed and the support-DNA complexwas washed with 1 mL water three times. One mL of 3.0M sodium acetatesolution was added to the complex and the tube rocked for a further 60minutes. After sedimentation, the supernatant was transferred to anothertube to which was added 2.5 mL of ethyl alcohol. The tube wascentrifuged at 13,000 ×G for approximately 5 minutes, during which timethe precipitate was sedimented.

After removal of the supernatant, the pellet was dissolved inapproximately 20 μL of a 20 mM tris buffer, pH 8.0. This solution, alongwith samples of the original DNA and a DNA-histone complex in solution,were applied to a 0.7% agarose gel and electrophoresed by conventionalmethods. The gel was stained with ethidium bromide to visualize themigrated DNA. The position of the fluorescent band from the pellet,compared to that of the unprocessed DNA sample demonstrated that the DNAadsorbed by the liquid affinity support of this invention was displacedby the sodium acetate treatment (without displacing the DNA-histonecomplex from the support) and could be recovered by ethanolprecipitation.

EXAMPLE 8 ANTIGEN CAPTURE AND RECOVERY

A perfluoroalkylated antibody, prepared as described in Example 4(A),was analyzed and was found to be 26% substituted. Aperfluorodecalin-based liquid affinity support was prepared by themethod described in Example 4(B).

A solution of radioactive human IgG was prepared by adding 10 μL of ¹²⁵I human immunoglobulin containing 1.5 μCi (New England Nuclear, Boston,Ma.) to 1 mL of a 1 mg/ml solution of human IgG (Jackson ImmunoResearchLabs. Avondale, Pa.) in 0.1M sodium phosphate, pH 8.0 (PB). Fifty μL ofthis solution was added to 1 mL of PB containing 250 μg of goat IgGfollowed by 50 μL of the support.

The reaction mixture was mixed by continuous rotation of the containerfor thirty minutes. The mixture was allowed to settle, the supernatantwas discarded and the sedimented support was washed three times with 1mL of PB and counted in a gamma counter to determine captured boundantigen (human IgG). After counting, 1 mL of 1.0M sodiumcarbonate/hydroxide buffer solution, pH 11.6, was added and the mixturemixed by rotation for 30 minutes. The mixture was allowed to settle, thesupernatant was discarded and the sedimented support was washed threetimes with 1 mL of PB and counted to determine remaining antigen. Theprocess of antigen capture and removal was repeated in a second cycleutilizing the recovered support. The results were as follows:

    ______________________________________                                        CPM                                                                           CYCLE   AFTER CAPTURE   AFTER REMOVAL                                         ______________________________________                                        1       10554            8330                                                 2       12985           10081                                                 ______________________________________                                    

These data demonstrate that the liquid affinity support of thisinvention is capable of capturing antigens and that such an IgG can bedisplaced from the support without displacing the capturing antibody.

EXAMPLE 9 PREPARATION AND USE OF A LIQUID CATION EXCHANGE SUPPORT

A cation exchange support was prepared by mixing 2 mL ofperfluorodecalin, 20 mL of deionized water, and 4 mL of Zonyl® FSP, ananionic fluorosurfactant, in a 50 mL polypropylene centrifuge tube. Thismixture was agitated vigorously on a vortex-mixer for 10-15 seconds, andthen centrifuged at approximately 1000 rpm for 3-5 minutes. The aqueouslayer was removed and 20 mL of deionized water was added to theemulsion. The emulsion was mixed on the vortex-mixer and centrifuged asdescribed above. This washing procedure, which removes the excessfluorosurfactant, was repeated three times.

This cation exchange support emulsion was used to extract a positivelycharged dye from an aqueous solution. One milliliter of a methylene bluesolution (8.8 mg/L) was added to one milliliter of the emulsion. Themixture was vortexed for several seconds and the emulsion was allowed tosettle for several minutes. The aqueous phase was removed and theabsorbance was measured spectrophotometrically at 666 nm. The absorbanceof the initial dye solution was 1.617; it decreased to 0.0118 after theextraction, demonstrating the successful removal of 99% of the dye witha liquid ion-exchange support of this invention.

EXAMPLE 10 PREPARATION AND USE OF AN ANION EXCHANGE SUPPORT

An anion exchange support emulsion was prepared as described in Example9, using the same quantities, except that Zonyl® FSC, a cationicfluorosurfactant, was substituted for Zonyl® FSP.

This emulsion was used to extract a negatively charged dye from anaqueous solution. One milliliter of a cresol red solution (12 mg/L in0.050M NaH₂ PO₄ solution) was added to one milliliter of the emulsion.This mixture was vortexed for several seconds and the emulsion wasallowed to settle for several minutes. The aqueous phase was removed andthe absorbance was measured spectrophotometrically at 434 nm. Theabsorbance decreased from 0.6250, for the initial dye solution, to0.0122 after extraction, indicating that 98% of the dye had beenremoved.

Approximately 74% of the dye was successfully recovered from the supportby washing it four times with one-milliliter portions of a solutioncontaining 1.0M NaCl and 0.050M NaH₂ PO₄.

EXAMPLE 11 SEPARATION OF DYES USING A CATION EXCHANGE SUPPORT

Two milliliters of a purple aqueous solution (pH adjusted to 8 withNAOH) containing methylene blue (approximately 9 mg/L) and cresol red(approximately 12 mg/L) was added to two milliliters of the cationexchange emulsion, which was prepared as described in Example 9. Thismixture was vortexed vigorously and the emulsion was allowed to settlefor several minutes. The lower perfluorocarbon phase became blue,leaving the red colored dye in the aqueous layer, demonstrating theseparation of oppositely charged species by liquid ion-exchange supportsof this invention.

EXAMPLE 12 EXTRACTION OF A PROTEIN USING A LIQUID ANION EXCHANGE SUPPORT

An anion exchange support was prepared by adding 20 mL of deionizedwater, 20 mL of perfluorodecalin, and 50 μL of a 1:4 mixture of Zonyl®FSN, a neutral fluorosurfactant, and Zonyl® FSC, a cationicfluorosurfactant, to a 125-mL separatory funnel. Zonyl® FSN was used tostabilize the emulsion and to reduce nonspecific adsorption of proteins.The mixture was shaken gently and allowed to settle for 5 minutes. Threelayers were formed in the separatory funnel. The bottom layer,containing the liquid support, was removed and retained and the middle(aqueous) phase was discarded. To the remaining foam (top layer),deionized water was added and the resulting perfluorocarbon supportemulsion was combined with the initially retained support. This waswashed twice with equal volumes of water.

Two milliliters of a 10 mM, pH 6.0 phosphate buffer containing 50 μg/mLof fluorescein-labeled human serum albumin (FITC-HSA) was added to twomilliliters of the anion exchange support emulsion. The mixture wasvortexed vigorously and the emulsion was allowed to settle for severalminutes. The aqueous layer was removed and the emulsion was washed fourtimes with 2-mL portions of the buffer solution. A small portion of theemulsion was removed and examined under a fluoroescence microscope.Fluorescence was observed on the surface of the droplets, demonstratingthe presence of the labeled HSA. No fluorescence was observed on thesurface of the droplets when the experiment was repeated with a neutralemulsion prepared with Zonyl® FSN in the absence of Zonyl® FSC.

The experiment described above was repeated and any fluorescenceremaining in the aqueous phase after the first extraction with theion-exchange support was measured with a fluorometer. This measurementindicated that 96% of the protein had been removed from the aqueousphase. 75% of the FITC-HSA could be recovered successfully by extractingfour times with two milliliter portions of a 0.25M NaH₂ PO₄ solution. Atthese same conditions, only small amounts (<25%) of fluorescein labeledIgG (FITC-IgG) was extracted, demonstrating the feasibility of proteinseparation using liquid ion-exchange supports of this invention.

EXAMPLE 13 CAPTURE OF IqG

A modified Protein A was perfluoroalkylated by dissolving 25 mg of arecombinant modified Protein A (expressed in E. Coli and lackingC-terminal domain, obtained from Porton Products Ltd. U.K.) in 25 mL of0.10M phosphate buffer, pH 8.5 (PB). Next, 0.5 mL of a 0.5% (v/v)solution of perfluorooctylpropyl isocyanate in tetrahydrofuran (THF) wasadded. After the addition of an additional 4.5 mL of pure THF, thereaction mixture was mixed gently and allowed to react for 2 hours. Thereaction mixture was then centrifuged at 3,000 rpm for 5 min. to removeany denatured protein or other insoluble reaction by-products.

A liquid affinity support was prepared by adding 100 μL ofperfluorodecalin, 100 μL of 0.10M phosphate buffer, pH 8.5, and 1.0 mLof perfluoroalkylated modified Protein A (prepared above) to each offour 1.5-mL micro centrifuge tubes. The tubes were shaken and thenvortexed. Next, 28.9 μL of 1.0, 0.1, 0.001 and 0% (w/v) solutions,respectively, of a blocking agent in water were added to each of thetubes and the tubes were again shaken and vortexed. The emulsion formedexhibited acceptable stability, except when no blocking agent wasutilized.

The blocking agent was prepared by first preparing F(CF₂)₆ CH₂ CH₂ OH.This alcohol, in turn, was prepared by reacting F(CF₂)₆ CH₂ CH₂ I withsulfuric acid followed by hydrolysis to give the alcohol. The iodide wasobtained by distillation of Zonyl® TelB fluorochemical intermediate (aregistered trademark of E. I. du Pont de Nemours and Company). Theblocking agent was prepared by reacting the alcohol (prepared above)with ethylene oxide in the presence of a mixed catalyst (NABH₄ and NaH)at 110° C. The ethylene oxide was bubbled through the reaction mixtureuntil the equivalent of an average of 14 units of ethylene oxide hadbeen added to the alcohol. The reaction mixture was then neutralizedwith acetic acid. The blocking agent so produced has the averagestructure:

    F(CF.sub.2).sub.6 (CH.sub.2).sub.2 --O--(CH.sub.2 CH.sub.2 O).sub.14 H

In order to test the ability of the modified Protein A-LPF affinitysupport to capture IgG (for subsequent removal of the purified IgG),several aliquots, ranging from 20-50 μL, of a 1.0 mg/mL solution ofFITC-IGG (human) was added to the tube and the tube was shaken andvortexed. The supernatant was removed and 100 μL of PB was added to washthe support; the tube was shaken and vortexed, and the supernatantremoved. This washing step was repeated twice with 50 μL of PB. Theaffinity support was then viewed under a fluorescence microscope. In allcases, fluorescence was observed on the surface of the droplets(support) indicating that the modified Protein A on the surface of thesupport was active and able to bind IgG. When treated with a glycine-HCIbuffer (pH 3). the IgG was removed into the aqueous portion as shown bytotal protein assay and fluorescence measurements.

We claim:
 1. A bioaffinity separation process comprising the stepsof:(A) forming a liquid affinity support by attaching a protein havingavailable amino groups to the surface of the droplets of an emulsion ofa liquid perfluorocarbon carrier through a highly fluorinated isocyanateanchor group by placing said anchor groups on about 20% of the availableamino groups of said protein; and (B) capturing a target ligand orbinder for the the ligand, complementary to the protein attached to thecarrier from a mixture using said liquid affinity support.
 2. Theprocess of claim 1 wherein said ligand is selected from the groupconsisting of enzyme substrate and enzyme inhibitors.
 3. The process ofclaim 1 wherein said binder for the ligand is selected from the groupconsisting of antibody, enzyme, and binding protein.
 4. The process ofclaim 1 wherein said ligand is an antigen.
 5. The process of claim 1wherein said ligand is a hapten.